About 20 million Americans have kidney disease. The number of people diagnosed with kidney disease has doubled each decade for the last two decades impacting on human suffering and enormous Medicare costs imposed by end-stage renal failure. Renal ultrafiltration is located within the renal glomerulus and performed by highly specialized podocyte cells. Podocyte foot processes (FPs) and the interposed slit diaphragms (SDs) cover the outer aspect of the glomerular filtration barrier and form a final barrier to protein loss. Damage of podocytes results in proteinuria and may lead to progressive decline of renal function. It is therefore in the interest of the public health to define the regulation of podocyte structure and function at the cellular and molecular levels and identify molecular targets involved in early structural changes leading to podocyte damage and the development of proteinuria. We have made the novel finding that cathepsin L enzyme is present within the cytoplasm of podocytes during nephrotic syndrome and cleaves CD2AP, an important SD protein. Cathepsin L is a lysosomal protease that has a broad biological significance such as intracellular protein degradation, activation of enzyme precursors, and tumor invasion. Normally located in lysosomes, cathepsin L can be found in the cytoplasm or can be secreted under certain conditions. We have developed a novel murine model of transient nephrotic syndrome that shares key features with human nephrotic syndrome which allows us to delineate proteolytic processes in podocytes resulting from the induction of cathepsin L. We propose 3 Specific Aims to unravel the function of extralysosomal cathepsin L expression and activity in podocytes. In the first Specific Aim, we propose to analyze the course and severity of proteinuria in mice lacking cathepsin L and how the lack of this enzyme affects CD2AP binding interactions with other SD proteins. We will then test the hypothesis in detail that the SD protein CD2AP is a proteolytic target protein of cathepsin L and explore the cell-junction stability of cathepsin L cleavage resistant CD2AP mutants (Specific Aim 2). In Specifc Aim 3, we will address the biological significance of cleaved CD2AP fragments and analyse the effects of such peptides on the actin based lysosomal degradome using organellar proteomics. The proposed role of proteolytic processing of podocyte structural and regulatory proteins during nephrotic syndrome represents a novel concept in the molecular work-up of proteinuria. If our hypothesis is correct, our work will have broad significance for the basic understanding of glomerular pathology e.g. the mechanism of podocyte FP effacement. Uncovering the role of podocyte proteolysis will help to develop novel pharmaco-therapeutics (such as cathepsin L resistant CD2AP mutants or small molecules blocking CD2AP cleavage) to tackle proteinuria and progression of glomerular disease. PERFORMANCE SITE(S) (organization, city, state) Massachusetts General Hospital, Boston, Massachusetts Harvard Medical School- Partners Healthcare Center for Genetics and Genomics PHS 398 (Rev. 09/04) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): REISER, Jochen KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Reiser, Jochen, MD PhD JREISER Mass General Hospital PI Sarracino, David, PhD Mass General Hospital Co-Investigator OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Alexander Y Rudensky, Ph.D. University of Washington Collaborator Thomas Reinheckel, M.D., Ph.D. University of Freiburg Collaborator Andrey S Shaw, M.D. Washington University Collaborator Human Embryonic Stem Cells Kl No Q Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.gov/reaistrv/index.asp.Usecontinuationpagesasneeded. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. Q Yes O No PHS 398 (Rev. 09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): REISER, Jochen The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description, Performance Sites, Key Personnel, Other Significant Contributors, and Human Embryonic Stem Cells 2-3 Table of Contents , Detailed Budget for Initial Budget Period (or Modular Budget) 5-6 Budget for Entire Proposed Period of Support (not applicable with Modular Budget) Budgets Pertaining to Consortium/Contractual Arrangements (not applicable with Modular Budget) Biographical Sketch - Principal Investigator/Program Director (Not to exceed four pages) 7-9 Other Biographical Sketches (Not to exceed four pages for each - Seeinstructions) 10-20 Resources 21-23 Research Plan 24-57 Introduction to Revised Application (Not to exceed 3 pages) 24-26 Introduction to Supplemental Application (Not to exceed one page). A. Specific Aims 27_ B. Background and Significance 28-31 C. Preliminary Studies/Progress Report/ (Items A-D: not to exceed 25 pages*) 31-41 Phase I Progress Report (SBIR/STTR Phase II ONLY) * SBIR/STTR Phase I: Items A-D limited to 15pages. D. Research Design and Methods 41-51 E. Human Subjects Research 52 Protection of Human Subjects (Required if Item 4 on the Face Page is marked Yes) Data and Safety Monitoring Plan (Required if Item 4 on the Face Page is marked Yes and a Phase I, II, or III clinical trial is proposed) Inclusion of Women and Minorities (Required if Item 4 on the Face Page is marked Yes and is Clinical Research) Targeted/Planned Enrollment Table (for new and continuing clinical research studies) Inclusion of Children (Required if Item 4 on the Face Page is marked Yes) F. Vertebrate Animals 52-53 G. Literature Cited 53-57 H. Consortium/Contractual Arrangements 57_ I. Resource Sharing 57_ J. Letters of Support (e.g., Consultants) 57 Commercialization Plan (SBIR/STTR Phase II and Fast-Track ONLY) Checklist Appendix (Five collated sets. No page numbering necessary for Appendix.) Check if Appendix is Appendices NOT PERMITTED for Phase I SBIR/STTR unless specifically solicited.... Included Number of publications and manuscripts accepted for publication (not to exceed 10) Other items (list): PHS 398 (Rev. 09/04) Page 4 Form Page 3